Clinical and Radiographic Evaluation of Applying Atorvastatin 1.2% Bio Adhesive with Plasma Rich in Growth Factor (PRGF) for Treatment of Mandibular Class II Furcation Defects: a Randomized Clinical Trial

Statement of the Problem: Molar teeth with furcation involvement are one of the most common problems in patients with periodontal disease. Regeneration methods are of the most controversial treatment strategies for these lesions. Purpose: The purpose of this study was to determine the effect of plasma rich in growth factors (PRGF) with 1.2% Atorvastatin (ATV) in the treatment of furcation involvement of mandibular molars. Materials and Method: The present randomized clinical trial was conducted on 15 patients with moderate periodontitis and class II furcation involvements; 24 defects were located in four groups of six, including debridement, ATV1.2%, PRGF, PRGF with ATV1.2%. The parameters of vertical probing depth (VPD), vertical clinical attachment level (VCAL), gingival index (GI), horizontal probing depth (HPD) and gingival recession (GR) were measured at baseline (T0), immediately before surgery (T1), 3 (T2), and 6 (T3) months after surgery. Moreover, the bone conditions were evaluated by digital subtraction radiography before and six months after surgery. Data were analyzed using SPSS23 software. Results: No significant difference in radiographic parameters was observed among the groups (p= 0.08). There was no significant difference in the mean levels of VPD, VCAL and HPD among the groups at different times (p<0.05). Comparison of clinical parameters of VPD, VCAL and GI in the treatment groups compared to the baseline showed a significant improvement in each group (p< 0.05) but there was no significant difference among different groups (p< 0.05). Conclusion: The use of PRGF with ATV 1.2% in grade II furcation involvement in mandi-bular molars was effective in the improvement of clinical and radiographic parameters six months after treatment, but this effect revealed no difference in comparison with the other groups.


Introduction
Periodontal disease is a multifactorial condition that causes pocket formation, clinical attachment loss, and bone resorption. Furcation involvement is defined as the destruction of periodontal tissue in the interradicular area of bone in multirooted teeth that occurs due to plaque-associated periodontal diseases [1][2][3]. Some of the features of furcation defects clinically include attachment loss in both vertical direction along the root and horizontal direction to the interior of the furcation area [4]. Concerning the complex morphology of the region, the furcation involvements is considered as one of the most challenging aspects of periodontal treatment and causes problems in the success of periodontal treatments [5][6].
Although the prognosis of furcation-involved molars has not been reported to be hopeless, the presence of periodontal disease in these areas will significantly increase tooth loss due to limited access to the dentist and patient. The treatment of periodontal lesions in these areas is one of the most difficult problems facing general dentists and periodontists, and so far, no completely predictable and ideal treatment has been provided for this condition [7].
In most cases, the outcome of the treatment is subjected to variety of causative factors including toothrelated (anatomical aspects) and patient-related factors (such as diabetes, smoking, stress, and so on) [2].
The treatment of periodontal furcation lesions in multi-root teeth is a major challenge in regenerative medicine [6-7, 12, 16]. The goal of periodontal treatment is to regenerate the tissues lost caused by periodontal disease. Lesions of the periodontal ligament and surrounding alveolar bone may result from infections of the periodontium or tissue of the dental pulp [17].
Many approaches to regenerative periodontal therapy are based on the use of growth factors and bone regeneration materials [7][8][18][19], guided tissue regeneration [7,17,20] and enamel matrix derivatives [20], in grade II furcation involvements. Statins are one of the materials that have been tested for the regeneration therapies [21][22].
Biologically active endogenous proteins offer a novel advance to tissue regeneration. In 1999, Anitua [23] proposed a technique for preparing plasma rich in growth factors (PRGF). This autologous preparation is enriched with biological mediators that accelerate regeneration of both hard and soft tissues. PRGF con-tains a high concentration of a platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor and has the least concentration of proinflammatory interleukins considering the absence of leukocytes [16].
The PRGF is a second-generation system, similar to platelet-rich plasma (PRP), which is used to obtain plasma proteins and platelets and requires less venous blood, is safe and easier to use, takes less time to prepare, and leads for faster healing [24]. It is reported that periodontal pocket improvement is related to increasing gingival epithelial attachment on the root surface through cell differentiation and proliferation. The growth factors released from the PRGF induce some bioprocesses such as cell proliferation, migration, and differentiation [24].
Mansouri et al. [6] employed bovine porous bone mineral plus PRGF for the treatment of grade II furcation and reported a significant reduction in the relative vertical clinical attachment level, horizontal clinical attachment level, and gingival index to baseline.
Statins are categorized as one of the lipid lowering drugs, which help reduce cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Moreover, Statins help promote angiogenesis and inhibiting metastasis by increasing the production of BMP-2 and hence, assisting in osteoblastic differentiation. They have different functions such as antiinflammatory, immune-modulatory, antioxidant, antithrombotic and endothelium stabilization roles. Osteoblastic differentiation and anti-inflammatory are those actions of statins, which can be employed to treat periodontal disease [25][26][27].
Atorvastatin (ATV ) has favorable effects on alveolar bone loss and tooth mobility and improves clinical parameters like probing depth reduction and clinical attachment level (CAL) gain when used as an adjunct to scaling and root planning (SRP) in the treatment of class II furcation defects [21][22].
To the best of authors' knowledge, no study has evaluated the effect of ATV gel combined with PRGF in the treatment of class II furcation lesions yet. Therefore, the present study was conducted to assess the effect of combined PRGF and ATV 1.2% on the treatment of furcation involvement in first and second molars of mandible.

Materials and Method
The current study was approved by the Ethical Committee of University (MUBABOL. REC.1396.25). It is also registered in the WHO clinical trial registry, branch of the Islamic Republic of Iran (IRCT: 20100427003813 N7) and a written informed consent was obtained from all subjects after providing a complete description of the study interventions.

Study design and eligibility criteria
This study was planned as a randomized, double blind Iran) three times a day for 10 days [6]. The sutures were removed after 14 days. In the follow-up sessions (2,4,6 months after surgery day), the supragingival plaque was removed if present and oral hygiene training was readministered as needed.

Clinical parameters
The clinical parameters were measured in all groups using the Williams probe (HU-Friedy; Chicago; IL; USA). These parameters included vertical clinical attachment level (VCAL) [the distance from the cementoenamel junction (CEJ) to the pocket depth], the vertical probing depth (VPD) (the distance from the free gingival margin to the pocket depth) [28], gingival index (GI) (based on Silness and Loe index) [29], recession depth (REC) (distance from the CEJ to the free gingival margin measured in midbuccal) [7], horizontal probing depth (HPD) (horizontal penetration of periodontal probe in furcation area) [8]. The VPD, VCAL, GI, HPD, and REC were recorded at baseline (T0), the time of surgery (T1), 3 months (T2), and 6 months after surgery (T3).

PRGF preparation
The preparation of PRGF was performed immediately before surgery, according to Anitua [30]. Thus, the blood sample (20 ml) was taken from all patients before surgery, poured into 5-ml test tubes containing an anticoagulant (3.8% sodium citrate), and then centrifuged at (buffy coat layer) containing white blood cells (0.5 ml); and layer 5 containing red blood cells. A 500-µl pipette was used to take both layers 1 and 2; a 100-µl pipette was used to take the PRGF layer in five small aliquots to avoid mixing with the layer 4. Then, 1 ml of PRGF was added by 50µl of calcium chloride (10%), thereby activating the plasma rich in growth factor.

ATV1.2% bio adhesive preparation
The biofilm or mucoadhesive patches were prepared by adding 95 cc of distilled water to 500cc of Erlenmeyer located on a warm plate stirrer. Next, the essential Carbopol [31] was added to water and heated. Afterwards, the methyl paraben and propyl paraben were solved in 95% of alcohol and then all solutions were added to the beaker as well. Next, the required amount of Glycerin was added to this solution. Finally, to prepare the bio adhesive, 0.5g of ATV per 100ml of gel was added [32].

Radiological assessment
The first radiograph was provided by using a pho-

Radiological findings
Interpretations of radiographs are shown in Table 1. The changes in radiopacity were found in two cases of ATV with PRGF group, and the Chi-square test showed no difference among the study groups (p= 0.08). Tables   2, 3 and 4. There was no significant difference in the mean VPD, VCAL and HPD among the studied groups at T0, T1, T2 and T3 (Tables 2 to 4). At T2, the GI score    Table 4).

Data of patients' clinical parameters are given in
Regardless of the evaluation time, the mean HPD level was not significantly different among the four groups and the interaction between the HPD level and the group had no significant difference (p> 0.05).  The repeated measurement ANOVA test revealed a statistically significant difference (p< 0.001) in the GI score among all groups from the baseline to the end line of the study (Table 5). In addition, regardless of the evaluation time, the mean GI score showed a significant difference among the four groups (p< 0.001), but the interaction between the HPD level and the group had no significant difference (p> 0.05).
The REC parameter was compared among four study groups from T0 to T3 using Fisher's exact and Friedman tests (p> 0.05) ( Table 6).

Discussion
In the present study, we conducted the clinical and radi-  However, the difference between the groups was not statistically significant. This finding showed that the increase in attachment plays a major role in reducing the probing depth in both groups and the change in gingival margin position is a small part of the decrease. Attachment increase can be due to true periodontal regeneration or defect healing by new connective tissue attachment or long junctional epithelium [8]. Histological studies are needed to determine the nature of attachment enhancement. Furthermore, the decrease in VPD in the ATV group may be due to the fact that statins inhibit inflammatory cells and MMP levels, which related to PPD and bleeding during probing and play an important role in the regeneration of connective tissue in periodontal disease [25].
The positive role of statins in periodontal regenerative therapy has been shown in systematic reviews [35][36]. Similar to the present research, Shirke et al. [25] and Pradeep et al. [37] evaluated the effect of ATV 1.2% on the treatment of chronic periodontitis, finding that ATV could decrease PPD and CAL when concurrently used with SRP.
In the present study, we evaluated the effect of the simultaneous use of ATV and PRGF Combination of these two substances showed synergistic effects in partial resolution of lesions on radiological exams in two out of the six samples, which was not statistically significant. This difference might be due to the small sample size recruited in the current research. We treated early grade II furcation defects and assumed that the remaining surrounding living tissues were able to provide a sufficient amount of periodontal target cells to be affected by growth factors present in PRGF and ATV.
Future studies on larger sample sizes along with open flap debridement are recommended.

Conclusion
Considering the limitations of the present study, PRGF along with ATV 1.2% was effective in the treatment of furcation defects in the mandibular molars and decreased GI, VPD and VCAL. However, there was no significant difference between this treatment modality and the other examined therapeutic items.